1. Field of the Invention
The present invention relates to a novel polyacrylamide gel and process for the preparation thereof. The polyacrylamide gel improves silver stain methods for detecting biomaterials, such as proteins, polypeptides, nucleic acids, and the like, which had been separated by electrophoresis procedures. Particularly, the novel polyacrylamide gel provides for the detection of proteins by ammoniacal silver staining with reduced background staining.
2. Discussion of Related Art
In the past, various methods have been used for protein analysis. Such methods include the Kjehdahl method, a colorimetric method, and others. In more recent times, electrophoresis techniques were employed. For example, a protein sample was fixed onto a matrix, such as a polyacrylamide gel, and subjected to electrophoretic separation. This separation is accomplished by exposing the sample to an electric field which causes the various components of the sample to migrate at different rates within the matrix These different rates are dependent on the charge of the individual components, as well as on the other physical and chemical properties thereof. Following the migration, a certain migration pattern is formed. Various methods for defining such migration patterns have developed, such as visual determination methods. Radioautography and staining are among the procedures which are included within the visualization methods.
Radioautography is conducted, for example, with intrinsically labelled proteins produced from substrates containing radioactive labelled amino acids. See article by P. H. O'Farrell, in J. Biol. Chem., Vol. 250, pp. 4007-4021 (1975). Moreover, extrinsic radioactive labelling techniques, such as iodination, can alter the electrophoresis properties of proteins, and the isotope is not necessarily uniformly linked to each protein and non-selectively distributed among all polypeptide components of the mixture.
Direct iodination of separated proteins in polyacrylamide gels has been reported by Edler et al. (see the article by J. H. Edler, R. A. Peckett, II, J. Hampton, and R. A. Lerner in the J. Biol Chem., Vol. 252, pp. 6510-6515 (1977)). This technique is quite useful when used to study radioactive peptides after tryptic digestion, but it has been found that some lots of acrylamide contain a contaminant which also becomes radioiodinated, and this negates its utility for radioautography.
Even though radioautography is a powerful visualization tool, it has certain disadvantages. For example, it is slow and complex. Also, it involves modifications to the proteins prior to electrophoresis.
One of the methods known for visualization of protein is described in U.S. Pat. No. 4,405,720. The stain described in this patent requires the use of three solutions and it takes a minimum of about 30 minutes to perform. Furthermore, the technique described in said patent does not stain proteins or nucleic acids in thin membranes such as cellulose nitrate.
Coomassie blue stain, the most commonly employed protein stain, takes hours to perform and it lacks the sensitivity to detect proteins present in low concentrations in biological fluids or tissues. Sensitivities achieved with heavy metal stains or fluorescent stains, on the other hand, were found to be less than, or at best, equivalent to Coomassie blue (about 10 ng of protein). Merril et al., Electrophoresis, 1982, pp. 327-342. Recently, more than a hundred-fold increase in sensitivity over that obtained with Coomassie staining was achieved by adapting a histological silver tissue stain for use with polyacrylamide gels. de Olmos, Brain, Behav. Evol. 2, 313-237 (1969), Switzer, et al. Anal. Biochem. 98, 231-237 (1979), Merril, et al. Proc. Natl. Acad Sci. U.S.A. 76, 4335-4339 (1979). This stain could detect as little as a tenth of a nanogram of protein and an image could be achieved in less than 6 hours.
There are a number of known methods useful in staining proteins which utilize silver. For example, L. Kerenyi, et al., Clin. Chim. Acta 38, 465-467 (1972), describes a method for demonstrating proteins in electrophoretic, immunoelectrophoretic and immunodiffusion preparations, whereby the preparations are treated with potassium ferrocyanide, which is transformed during development into silver ferrocyanide and then into colloidal silver grains. The physical developer contains anhydrous sodium carbonate, ammonium nitrate, silver nitrate, tungstosilicic acid and formalin, and the protein in the preparations stain dark brown with a page gray background.
R. C. Switzer, et al., Anal. Biochem. 98, 231-237 (1979), and C. R. Merril, et al., Proc. Natl. Acad. Sci. U.S.A., 76 No. 9, 4335-4339 (1979), describe a silver stain technique for detecting proteins and peptides in polyacrylamide gels which is a modification of de Olmos' neural, cupric-silver stain. The procedure consists of ten steps and utilizes an aqueous solution of silver nitrate and cupric nitrate and involves treatment with a diamine solution, which is known to sometimes form an explosive silver amide complex. The proteins stain as dark spots on a darkened background.
B. A. Oakley, et al., Anal. Biochem. 105, 361-363 (1980), simplified the above procedure of Switzer, et al., by reducing the number of steps involved to six and also reducing the amount of silver required without diminishing the sensitivity of the technique. However, the manner in which the proteins stain was not changed, i.e., dark stain on a darkened background.
A further modification of the Switzer, et al., procedure was made by R. C. Allen, Electrophoresis I, 32-37 (1980), who increased the sodium to ammonium ion ratio, which resulted in increased silver deposition.
C. R. Merrill, et al., Anal. Biochem, 110, 201-207 (1981), modified and simplified the above procedure of Kerenyi, et al., adapting it to acrylamide gels.
D. Goldman, et al., Clin. Chem. 26 No. 9, 1317-1322 (1980), report that when using a procedure essentially the same as that of Merrill, et al. (PNAS, 1976), and Switzer et al., (Anal. Biochem., 1979), proteins from samples of cerebral spinal fluid stained in shades of yellow, red and blue.
C. R. Merrill, et al., Science 211, 1437-1438 (1981), describe a silver stain procedure for proteins separated by two-dimensional gel electrophoresis, which requires treatment with potassium dichromate and nitric acid prior to staining with silver nitrate followed by washing and then immersion in an image developer containing formalin and sodium carbonate. There is no indication of color development with this stain procedure.
Poehling and Neuhoff, Electrophoresis 1981, 2, 141-147, describe a silver stain suitable for acrylamide gels of 0.5 to 1 mm thickness which requires a pretreatment with glutardialdehyde under controlled temperatures prior to staining with a diamine solution.
Marshall and Latner, Electrophoresis 1981, 2, 228-235, describe a silver stain method which requires a treatment with paraformaldehyde and sodium cacodylate prior to staining with a modified diamine solution wherein methylamine is substituted for ammonium hydroxide. Ochs et al., Electrophoresis 1981, 2, 304-307, and Sammons and Adams, Electrophoresis 1981, 2, 135-145, describe a silver stain procedure of which the present invention is a modification.
With the exception of the 1980 Goldman et al. procedure and the method of Sammons and Adams, all of the silver stain techniques described above only stain proteins in varying shades of brown or black.
U.S. Pat. No. 4,416,998 describes a silver stain procedure, wherein a substance capable of binding silver is treated with a glutaraldehyde solution, an aqueous silver salt solution, a reducing solution and an aqueous carbonate or sulfate solution. The procedure also enables one to stain a variety of substances, including protein, in varying shades of color.
U.S. Pat. No. 4,582,808 describes a silver staining method comprising pretreating a carrier, such as a polyacrylamide gel, with an alcoholic solution containing polyethylene glycol or polyoxyethylene alkylphenol, followed by treating the pretreated carrier with a solution of silver nitrate. This method is disclosed as having a shortened operation time and an improvement in the reproducibility of staining.
U.S. Pat. No. 4,555,490 describes a method using light ("photodevelopment") to develop a metallic silver image of biopolymers, particularly nucleic acids and proteins separated on polyacrylamide gels, whereby it is possible to visualize protein and nucleic acid patterns within 10 minutes after electrophoretic separation. This "photodevelopment" method requires only two solutions: a solution to "fix" the proteins and a solution containing silver ions, which produces an image when exposed to light. This type of protein stain has achieved a sensitivity of about 0.5 ng of protein. DNA separated on polyacrylamide may also be visualized with this stain.
U.S. Pat. No. 4,575,452 describes a method and kit for the optical detection of proteins and nucleic acids in a matrix, such as polyaorylamide electrophoresis gels. The method comprises fixing the proteins and nucleic acids in the matrix using aromatic sulfonic acids having tertiary amines capable of forming coordination complexes with silver ion.
U.S. Pat. No. 4,672,043 describes a method for determining macromolecules in polyacrylamide gels comprising the steps of forming a latent stain image by nucleating the macromolecules in the gel with a palladium tetramine salt and developing the latent stain image by treating the gel with a physical developing solution comprising dimethylamine borane and a transition metal salt. The improvement comprises contacting the developed latent stain image with a 1-phenyl-2-tetrazoline-5-thione or a salt of 1-phenyl-1H-tetrazole-5-thiol.
U.S. Pat. No. 4,690,901 describes a staining technique for specimens, which involves the sequential treatment of specimens with periodic or hydrochloric acid, thiocarbohydrazide or thiosemicarbazide, and silver methenamine. The technique, when using periodic acid, provides an excellent stain to evaluate glycomacromolecules and fibrovascular tissue and to conduct a broad spectrum of staining procedures for all modes of microscopy. Use of hydrochloric acid facilitates evaluation of cell nuclear DNA and chromatin.
U.S. Pat. No 4,695,548 describes gel inserts comprising a solidified liquid, such as agarose, suitable for use in an electrophoretic method, lysed cells entrapped within a matrix formed by the solidified liquid and macromolecules, such as DNA or intact chromosomes derived from the lysed cells, may be advantageously used in electrophoretic separations. The gel inserts are placed directly in a suitable support medium and subjected to one or more electric fields to separate the macromolecules.
U.S. Pat. No. 4,468,466 describes a silver stain method for protein in gels utilizing treatment with a reducing agent followed by treatment with a silver salt and actuating irradiation, the improvement comprising the use of a reducing agent consisting essentially of dithiothreitol in an amount effective to stain the protein but keep background staining to a minimum.
As illustrated above, silver staining methods, which employ polyacrylamide gel for detecting biomaterials, are widely used. However, unacceptable background staining drawbacks are associated with each of these illustrative methods, including the method of U.S. Pat. No. 4,468,466, which is mentioned as keeping background staining to a minimum.
Recent observations concerning the mechanisms of silver stains have led to the development of a polyacrylamide gel, which does produce very little, if any, background staining. The key observations, which permitted the development of this gel, are: the essential nature of basic amino acids containing sulfur in the detection of peptides by the silver staining reaction; and evidence that the active groups in the basic amino acids, the imidazole, guanidine and amino groups, or the sulfur groups in the sulfur containing amino acids, require cooperative effects. That is, they function poorly when they are isolated in a polymer, but if two or more basic amino acids of sulfur containing amino acids are in close proximity, then a good staining reaction will occur. These studies on the mechanisms of silver stains led the present inventors to determine that the amide groups in methylene-bisacrylamide crosslinking agent might be responsible at least partially for the background found with the silver stains. Methylene-bisacrylamide contains two amide groups, which are separated by a single carbon. In this study, the present inventors have demonstrated the role of these amide groups in the formation of the background stain by studying the silver stain reaction in gels containing varying ratios of acrylamide to the methylene-bisacrylamide crosslinking agent. By utilizing different crosslinking agents, the present inventors have demonstrated that the appearance of background staining depends mainly on the presence of and the position of the amido groups in the crosslinking agents. It depends also on the presence of other groups in the crosslinker or the acrylamide chain.